Ergebnisse der Mikrobiologie Immunitätsforschung und Experimentellen Therapie: Fortsetzung der Ergebnisse der Hygiene Bakteriologie · Immunitätsforschung und Experimentellen Therapie [1. Aufl.] 978-3-540-03126-0;978-3-662-42622-7

Table of contents :
Front Matter ....Pages i-iii
Recent advances in measles virology (Seiji Arakawa)....Pages 1-38
Advances in rabies research (Karl Habel)....Pages 39-54
Tric viruses: Agents of trachoma and inclusion conjunctivitis (Ernest Jawetz, Phillips Thygeson)....Pages 55-95
Virustropismus. Neue Erkenntnisse aus Untersuchungen über die Enterovirus-Infektion der Zelle (Leroy C. McLaren, Gerhard Brand)....Pages 96-115
Die interstitielle plasmacelluläre Pneumonie und Pneumocystis carinii (Wolfgang Bommer)....Pages 116-197
Oxygen-stable hemolysins of β-hemolytic streptococci (Isaac Ginsburg, T. N. Harris)....Pages 198-222
Grundzüge der Virusätiologie von Tumoren nach neueren Ergebnissen (Klaus Munk)....Pages 223-283
A Review of Recent Studies on Rous Sarcoma Virus (RSV) Emphasizing Virus-Cell-Host Interactions (Herbert J. Spencer, Vincent Groupé)....Pages 284-311
Back Matter ....Pages 312-351

Citation preview

ERGEBNISSE DER MIKROBIOLOGIE IMMUNITÄTSFORSCHUNG UND EXPERIMENTELLEN THERAPIE FORTSETZUNG DER ERGEBNISSE DER HYGIENE BAKTERIOLOGIE· IMMUNITATSFORSCHUNG UND EXPERIMENTELLEN THERAPIE BEGRUNDET VON WOLFGANG WEICHARDT

HERAUSGEGEBEN VON

W.HENLE

W.KIKUTH

K.F.MEYER

PHILADELPHIA

DUSSELDORF

SAN FRANCISCO

E.G. NAUCK

J.TOMCSIK

HAMBURG

BASEL

ACHTUNDDREISSIGSTER BAND MIT 43 ABBILDUNGEN

SPRINGER-VERLAG BERLIN HEIDELBERG GMBH 1964

ISBN 978-3-662-42623-4 ISBN 978-3-662-42622-7 (eBook) DOI 10.1007/978-3-662-42622-7 Alle Rechte, insbesondere das der Übersetzung in fremde Sprachen, vorbehalten Ohne ansdrückliehe Genehmigung des Verlages ist es auch nicht gestattet, dieses Buch oder Teile darans auf photomechanischem Wege (Photokopie, Jlfikrokopie) oder auf andere Art zu vervielfältigen

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Inhaltsverzeichnis Seite

Recent advances in measles virology. By Professor Dr. SEIJI ARAKAWA, TokyofJapan. With 3 figures . . . . . . . . . . . . . . . .

1

Advances in rabies research. By Dr. K. HABEL, Bethesda, Md.fUSA.

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Tric Viruses: Agents of trachoma and inclusion conjunctivitis. By Professor Dr. ERNEST JAWETZ, San Francisco, Calif.JUSA., and Professor Dr. PHILLIPS THYGESON, San Francisco, Calif.JUSA. . . . . . . . . . . . . .

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Virustropismus. Neue Erkenntnisse aus Untersuchungen über die Enterovirus-Infektion der Zelle. Von Professor Dr. LEROY 0. MoLAREN, Albuquerque, New MexicoJUSA., und Professor Dr. GERRAR BRAND, Minneapolis, Minn.JUSA. . . . . . . . . . . . . . . . . . . . . .

96

Die interstitielle plasmacelluläre Pneumonie und Pneumocystis carinü. Von 116 Dr. WoLFGANG BoMMER, MarburgJLahn. Mit 31 Abbildungen Oxygen-stable hemolysins of ß-hemolytic streptococci. By Dr. IsAAC GINSBURG, Philadelphia, Penn.JUSA., and Dr. T. N. HARRIS, Philadelphia, Penn.JUSA. With 6 figures . . . . . . . . . . . . . . . . . . . . 197 Grundzüge der Virusätiologie von Tumoren nach neueren Ergebnissen. Von Dr. KLAUS MuNK, Heidelberg. Mit 3 Abbildungen . . . . . . . . . . 223 A review of recent studies on Rous Sarcoma Virus (RSV) emphasizing viruscell-host interactions. By Dr. HERBERT J. SPENCER, Cooperstown, N.Y.J USA., and Dr. VINCENT GROUPE, New Brunswick, N.J.jUSA. 284 Namenverzeichnis

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Sachverzeichnis .

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Recent advances in measles virology By

SEIJI ARAKAWA With 3 figures Table of contents I.

II.

III. IV. V.

VI. VII.

Introduction . . . . . . . Inoculation of virus in animals and tissues a) Monkey. . . . . . . . . . . b) Mouse . . . . . . . . . . . c) Embryonated hen's egg culture d) Other animals . . . . . . . Tissue culture . . . . . . . . . a) Isolation of the measles virus . b) Passage of the measles virus in tissue culture systems c) Attenuated virus and virulent virus . . . . . . . . d) Spontaneaus agents similar to the measles virus in uninoculated cultures e) Appearance of multinuclear giant cells f) Inclusion bodies . . . . . . . . . Physical properties of the measles virus Relationship to other viruses. Vaccine . . . . . . . . . a) Prevention . . . . . . . b) Treatment with vaccine. . Measles immune serum and its fractions . Measles encephalitis and mouse brain fixed virus . Addendum. References . . . . . . . . . . . . . . . . . .

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Experimental work on Measles dates from 1911, when ANDERSON and GoLDBEBGER succeeded in transmitting the disease from man to the macaque monkey. Progress, however, was initially hampered by difficulty encountered in transmitting the illness to experimental animals other than mice. But since the successful isolation of the measles virus in human or monkey kidney cells by ENDERS and PEEBLES (1954), very promising advances in this field of research have been made by many workers.

I. lnoculation of the virus in animals and tissues a) Monkey NrcoLLE and CoNSEIL (1911), on studying the susceptibility of monkeys to measles, found that M. sinicus developed pyrexia only as a result of the intraErgebnisse der Mikrobiologie, Bd. 38

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peritoneal inoculation of patient sera. ANDERSON and GoLDBERGER (1911), on the other hand, having inoculated M. rhesus and M. cynomolgus with the blood and the buccal and nasal secretions of patients, were able to report the occasional development of a rash and other symptoms typical of human measles, as well as the successful serial passage of the virus through monkeys and the establishment of infection by contact. At the same time they discovered the blood of patients to be infective from just before till 24 hours after appearance of the rash, while the buccal and nasal secretions remained infective even for 48 hours. HEKTOEN and EGGERS (1911) inoculated M. rhesus with the blood of patients collected immediately after the appearance of the rash, and noted the development of symptomatic measles accompanied by a leukocytosis which was followed by a variable degree of leukopenia (mainly granulopenia with a slight relative lymphocytosis). Lucus and PRIZER (1912) were able to report the appearance of Koplik spots as weil as the development of pyrexia and rashes. A transient rise in the neutrophil count, and also that of large monocytes, may precede symptoms, and is said to be marked in the milder cases. TuNNICLIFF (1912) has found a fall in polymorphonuclear neutrophils and lymphocytes together with a very small increase in large monocytes. These observations, however, were confined to one single animal. JuRGELUNAS (1954), on the other hand, having repeated the work of ANDERSON et al. on a population of 10 monkeys (M. cynomolgus, rhesus and baboon) was sceptical regarding the susceptibility of these animals, because only one single baboon had developed a rash resembling that of human measles in any way. SELLARDS (1919) expressed similar views. He and WENTWORTH, having injected 3 animals with infected blood, found the monkeys to remain entirely free from any symptoms that were either diagnostic or even suggestive of measles. Retransmission to man was unsuccessful. Yet, NrcoLLE and CoNSEIL (1920) confirmed the susceptibility of monkeys, and did succeed in reinfecting man. In their view, the monkeys used by SELLARDS had probably already been infected with measles. BLAKE and TRASK (1921) injected the nasopharyngeal washings of patients into 10M. rhesus resulting in the development of symptoms closely resembling those of human measles in 8 of the animals. In addition, they were able to pass the virus through a series of experimental animals, and also to demonstrate the acquisition of immunity. KAwAMURA (1922) injected M. fuscatus with the blood of patients collected 60 hours before and immediately after appearance of the rash, and obtained a clinical picture almost identical to that of human measles, including Koplik spots, rashes, etc., the Koplik spots being observed in all of the 8 animals in the series. DEGKWITZ (1927), however, failed to produce the typical symptoms of measles in about 80 M. rhesus inoculated, while in subsequent experiments conducted in association with Mayer on 100 animals, the symptoms occurred in only 10% of the monkeys inoculated. From comparative studies of the disease in monkeys and man he concluded that the origin of spontaneaus infection could be traced even in the Rhesus monkey. KRAFT (1932) expressed doubt regarding the susceptibility of monkeys, because out of 34 rhesus monkeys only one developed pyrexia, leukopenia, Koplik spots and a rash of 2 days' duration, following the intramuscular, subcutaneous, intracardial, or intratracheal inoculation of the blood or nasopharyngeal secretions of patients collected immediately after appearance of the rash. TANIGUCHI

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Recent advances in measles virology

et al. (1954) reported that out of 99 monkeys (M. rhesus, M. cyclopis, M. irus, and M. fuscatus) inoculated, only 4 developed Koplik spots, and only 2 developed the rash. RAKE et al. (1939-1941) consistently found the rash in all positive cases, but Koplik spots only in rare instances, namely in 4 out of 11 cases inoculated with patient materials (1940), in 2 out of 20 cases inoculated with embryonated hen's egg material Day of illness (1940), and in none out I s 7 .9 II IJ IS 17 1.9 .?1 J of 11 cases inoculated ;"q~--t-:Jfrunlhema t- 1I ~i...i with tissue culture ma-"'"' i-1-. 80 1-' I I I I terial (1941). 8HAFFER •- • ~ukocyte r- ' ........ osinz,htl et al. (1941 ), having •-·-• tymp OCJ:'fe ---~- r,/1 •--• w P/onocy"'e t- inoculated batches of - · S/offce/1 Ir___. ~ 1\ M. mulatta with specifeulrop!Jtl 1- • ··•I!IJ.'&0 .\ ~ '\ mens from human and .?0 / /llfJ.'IJIJ I 11"1 \ chorio-allantoic cultures, IS v· 1'-., J'o' ~-'\ /(}()(}0 found enanthema closely ...., II ~ '._ .v-" 10 l.-.. -... - i\. ·• \1 / .~ resembling that of mea' .. otJ.'&IJ ... ?- ·• .. . .. ·· s J.-o· -· · sles developing in 48.1% .. r.. j'.' ' 'IJIJ (J - -1-o. . •· :t.l:-t.l::: :: .~t!'.-- .... of 35 andin 45.5% of 47 IJ monkeys, respectively. Fig. 1a. Results of inoculation with mouse adapted measles virus Ohki strain in Macacus irus (1955) HURSTandCooKE (1941 ), too, were able to induce symptoms of measles in M. mulatta, M. radiacus and Cebus fatuellus by means of various combinations of intramuscular, intranasal, intratesticular, intravenous, and intratracheal inoculations with patients' defibrinated blood and filtrates of nasal washings. These authors were apunsuccessful parently with M. irus, M. maurus, and Cynopithecus niger. Fig. 1b. Koplik spots 13 days after inoculation with Ohki strain (1955) ARAKAWA (1951 ), having detected a neutralising antibody of the virus in monkeys proving resistent to infection by direct inoculation, considered that these aninlals must in the past have contracted measles or a related disease. Accordingly, ARAKAwA et al. (1954), followed by SATO (1959), inj ected mouse fixed virus into 7 young monkeys (M. irus and M. fuscatus) considered not to have been exposed to infection and therefore to have no antibodies. All the monkeys, except the one selected for killing on appearance of the enanthema, developed rash, pyrexia and enanthema, and particularly Koplik spots, very much like those seeninhuman measles (Fig. 1). The average incubation period up to onset of the rash is 12 days or more, Koplik 0

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SEIJI ABAKAWA:

spots or enanthema appearing after 8 days. The white count rose in the initial stages of the incubation period and feil around the height of the illness. Conjunctivitis, rhinitis, and anorexia, too, were noted in the majority of cases, together with occasional complications such as diarrhoea and bronchitis. In monkeys killed during the prodromal stage, there was tissue darnage corresponding to the enanthema, and mononuclear infiltrations in the submucosa. Giant celllike granuloma formation was observed in the lymph follicles of the sublinguallymph gland, thought to correspond to the giant cells of Warthin-Finkeldey to be described later. PEEBLES et al. (1957) using tissue culture virus and complement fixing and neutralising antibody techniques, also arrived at the conclusion that monkeys were susceptible. However, of 10 monkeys, which had previously been proved tobe free from antibodies, only 5 were infected by the injection of tissue culture virus or patient's blood. The reason for this may well have been the small size of the doses injected. SERGIEV et al. (1960) conducted experiments on 14 Papio hamadryas and P. hybrides, ll-17 months old. They inoculated 4 of them with blood from a sick child in the prodromal stage, and collected specimens of blood from the 4 monkeys 8-9 days after inoculation, in order to estimate virus titre and antibody content with the aid of the AVB technique (agglutination of virus absorbed on bacteria). The 10 remaining monkeys were killed 3-17 days after inoculation for histopathological evaluation. In 8 of the monkeys, a rash was seen 8 and ll days after inoculation. The primary phase of viraemia occurs 5-6 days after inoculation, the virus being fixed to the reticulo-endothelial and lymphoid tissue where it proliferates. Proliferation in the lymphadenoid tissue leads to peculiar inflammatory lesions with specific giant cells, and results in a renewed rise of the virus level in the blood. Multinuclear giant cells are found as early as 3 days after inoculation, and characteristic giant cells are said to appear in the second stage of viraemia. Morphological changes in the skin and mucosa make their appearance at the end of the second stage of viraemia, be it haematogenic or lymphogenic. With the onset of the rash, the virus in the blood decreases or disappears while the antibody titre begins to rise and the lymphadenoid giant cells to disintegrate. RucKLE (1956, 1958) isolated from Cynomolgus monkeys a virus related to the human measles virus, which he asserted to be biologically, chemically, immunologically, and epidemiologically homologous to the human measles virus. SABURI et al. (1961) and ÜGIWARA et al. (1961) find that monkeys recently imported are entirely free from antibodies, whereas antibodies may be present in animals which have been reared in zoological gardens and laboratories. They consider that the infection must have been transmitted from man. MEYER et al. (1962) working with Indian Rhesus monkeys, made an ecological survey, and were able to confirm that monkeys in their natural habitat were free from the disease, but became infected on the way to captivity. PERlE and ÜHANY have reported (1960) that the blood cells of baboons (Cinocephales) yield haemagglutination and haemolysis reactions with measles virus, and that both reactions are inhibited by convalescent serum. Similar haemagglutination reactions were observed by RosEN (1961) in M. mulatta and by RosANOFF (1961) in the same species as well as the African green monkey (Ceropithecus aethiops). According to RosANOFF, baboon kidney propagated virus yielded higher levels of haem-

Recent advances in measles virology

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agglutinins than measles virus grown in any other primate tissue, while chick tissue propagated virus failed to produce any haemagglutinins. ARAKAWA (1962), too, has found haemagglutinins in Rhesus monkey, but less convincingly in M. irus and M. fuscatus.

b) Mouse GAVRILOV (1937) has studied the histopathological changes in the brains of mice, using a technique of injecting materials from measles patients intracerebrally. The susceptibility of mice to the measles virus has been demonstrated by YAor and ARAKAWA (1942). ARAKAWA (1948) found that measles virus from the serum of patients collected at the time of the prodromal fever prior to the appearance of Koplik spots, can be isolated and fixed in mouse brain. The same serum after passage through embryonated hen's eggs is fatal to mice when injected intracerebrally. In subsequent papers, he describes the immunological and serological identification of the virus (1949, 1954) as well as its successful transmission back to human subjects (1954). He reports the appearance of Koplik spots, rashes, pyrexia, etc. in both monkeys and man (1954). YAMADA (1951) demonstrated a rise in antibodies by means of neutralization and complement fixation tests, using serum from measles patients as well as mouse fixed virus. His findings have been confirmed by WADA and ÜGAWA (1955) and by FunTA (1955). ÜGAWA (1955) injected the strains isolated by WADA and by ARAKAWA into monkeys, guinea pigs and mice, and obtained histopathological findings similar to those revealed on post-mortem examination of children who had died of severe measles pneumonia, but different from those found in the organs of animals inoculated with the Japanese B encephalitis virus by way of control. He emphasises that in measles pneumonia it is unusual for cellular infiltration to be followed by exudation, interstitial proliferation being the characteristic change. Catarrhal pneumonia and catarrhal bronchitis occur only exceptionally, while interstitial pneumonia is the form commonly observed. The pneumonia due to Japanese B encephalitis, on the other hand, the catarrhal form as well as catarrhal bronchitis do occur beside interstitial pneumonia. However, no special attention has been paid to lymphadenoid cells and giant epithelial cells. IMAGAWA and ADAMS (1958) and CARLSTRÖM (1958) succeeded in isolating the measles virus in suckling mouse brain. SuzuKI et al. (1958), using Enders' technique, cultivated the mouse fixed virus in monkey kidney cells, and identified the strains serologically. Subsequently, H. TANIGUCHI (1959) operating with 41 specimens between 1952 and 1957, was able to obtain one strain from the blood specimens and one strain from spinal specimens from a patient with measles encephalitis, and to demonstrate the serological identity of these strains with Arakawa's strain. They found it possible to cultivate these viruses in such cells as human foetal tissue, human kidney tissue, monkey kidney tissue and pig foetal tissue. Morphological comparision with the ENDERS' Edmonton strain grown in monkey kidney cells, revealed the appearance of similar multinuclear giant cells, vacuoles and cytoplasmic and intra-nuclear inclusion bodies, as well as the presence of a similar antigen. In addition, he states that mouse fixed Ergebnisse der Mikrobiologie, Bd. 38 la

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virus which had passed through chick embryo tissue culture had lost much of its pathogenicity to mouse already after the third passage. ARAKAWA et al. (1959) repeated experiments designed to isolate the virus from young mice inoculated with specimens collected from patients during 1953 to 1957, and passaged through embryonated hen's egg chorio-allantoic membrane. Embryonatedhen's egg treated with salinewas inoculated into mice of the same litter as control, the results being entirely negative. They estimated complement fixation and neutralising antibody titres of sera collected between 1953 and 1957 from patients or their parents against the mouse fixed virus, and were able to demonstrate the development of antigens in the convalescent sera of measles patients, serially collected on several successive days. They also found that sera from some patients and inmates who had not developed clinical measles, showed gradually increasing titres for neutralisation and complement fixation against the mouse adapted measles virus. In addition, complement fixation tests using antigen prepared from the mouse adapted measles virus, were carried out on 107 measles patients on 3-5 different occasions at various intervals. 75 of these 104 subjects developed specific antibodies against measles, the antibodies generally making their appearance within 3 months of infection, and disappearing again between the 4th and 6th month. The intensity of complement fixationwas not found to be related to sex, but on classification by age was found to be less marked in children under 3 years of age. The antibody responses observed among members of the same family were usually similar. The complement fixing responses are apparently less marked than those obtained with tissue culture antigen. Thus, with the mouse adapted virus antigen, the antibody response is obtained within one month in 32% of 107 cases, andin 64% within 2 months, with a maximum titre of 1 : 64. With tissue culture antigen, the response occurs 7 days, (ENDERS and PEEBLES 1954), 24-69 hours (RucKLE and RoGERS 1957), and 2-40 days after appearance of the rash (GIRARDI et al. 1958), with titres as high as 1:512. Since a serum titre of 1 : 32 with the crude antigen is said by GIRARDI et al. to correspond to a titre of 1:128 with the 10 fold concentrated antigen, it will be necessary to use purified and concentrated mouse brain or hen's egg antigen. With regard to the neutralising antibody response, on the other hand, mouse brain shows a relatively higher titre. In the course of his work on the identification of the mouse adapted virus, ARAKAWA has established the prophylactic effectiveness of mouse brain vaccine (1949), and of vaccine prepared from embryonated hen's egg culture (1955). He also found that hyperimmune serum prepared with mouse adapted viruswas effective therapeutically. The reduced potency of a vaccine prepared in 1960 may have been due to contaminants. The cytopathic effects typical of the measles virus, on the other hand, have been demonstrated on inoculation into human amniotic cells of the ENDERS' Edmonton strain, after 8 passages through chorioallantoic membrane of embryonated hen's eggs, and 2 passages through mouse brain in vivo.

c) Embryonated hen's egg culture ToRRES and TEIXEIRA (1935) inoculated developing hen's egg with material from measles patients. The pocks which formed on the chorio-allantois consisted

Recent advances in measles virology

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of uctodermal proliferations with infiltration into the mesoderm. The formation of pocks on the chorio-allantoic membrane have also been reported by WENCKEBACH and KUNERT (1937}, DEGKWITZ and MAYER (1937), HEINZMANN (1939), RITOSSA and MULE (1941}, and NISIKI (1942). ARAKAWA (1948) reported similar findings but according to RAKE and SHAFFER (1939, 1940) the changes arenot specüic. RAKE and SHAFFER (1940) transmitted material from egg culture to monkeys, and SHAFFER et al. were able to produce the symptomsofamild case of measles by transmitting the material to both monkey and man. HuRST and CooKE (1941) and MüLLER (1942) were unable to cultivate the measles virus on chorio-allantoic membrane and amniotic cavity. ARAKAwA (1949), and ARAKAwA et al. (1954) successfully transmitted material which had been grown on embryonated hen's egg to mice, which developed the illness and died. They reported on the prophylactic effectiveness of a vaccine prepared from embryonated hen's eggs inoculated with this mouse adapted virus (1956). TANIGUCHI et al. (1956) transferred the blood and nasopharyngeal washings of measles patients and the blood of infected monkeys onto the chorio-allantoic membrane of embryonated hen's egg, but the material thus obtained displayed little or no pathogenicity for either man or monkey. They therefore considered the possibility of inducing proliferation of the virus on the chorio-allantoic membrane by means of interference with mumps virus, using the result of prophylactic inoculation in man as an indirect criterion. MILOVANOVI6 et al. (1957) tried to inoculate the virus by the chorio-allantoic membrane by the combined nse of tissue culture with negative results. However, they were able to repeat successfully the cultivation in the egg amniotic sac of a strain which had passed through human amniotic tissue. According to ÜKUNO etal. (1960}, the tissue culture strain can also be cultivated on the chorioallantoic membrane, although it is not cytopathic to human stable amnion FL cells after two or more passages. Interference with the mumps virus, however, does take place. The authors claim that the virus passed through amnion exerts a cytopathic effect as well as interference activity, and they were able to induce measles in man by means of nasal inhalation of the first generation chorio-allantoic passage of the amnion cultured virus. Inoculation measles, however, was not induced by second or subsequent generations of the strain, although a rise in antibody titre was noted in some cases. The transmission, by inhalation or injection of virus grown for two generations on chorio-allantois to monkeys led to a considerable rise in antibody titre without giving rise to any clinical manifestations. ÜKUNO also conducted a field experiment using virus grown on amniotic cavity and passing it through chorio-allantoic cavity for 2-3 generations. Although the strain proved noncytopathic to tissue culture, it induced a mild measles syndrome similar tothat caused by the ENDERS' attenuated virus. More recently (1962), he has been working on experimental prophylaxis with virus of 10 TCID 50 grown on egg amnion on a long term basis. ARAKAWA et al. (1962) have prepared both inactivated and live vaccines from hens egg chorio-allantoic membrane cultures of the Edmonton strain after 8 passages through chorio-allantoic membrane, 2 passages through mice and several passages through human amniotic cells. Injecting these vaccines subcutaneously, he was able to induce rises in antibody titre as well as to demonstrate their effectiveness in protecting against natural infection without giving rise to any

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reactions. According to ARAKAWA (1962), cytopathic changes in .FL cells do occur in response to virus passed through chorio-allantoic membrane.

d) Other animals .FRANKEL (1958) was able to produce the illness m suckling hamsters by intra-cerebral inoculation. A nurober of papers published deal with the susceptibility of rabbits and guinea pigs (VAN RooYEN and RHODES 1951), most of them reporting failure to infect these animals. Rahbits and guinea pigs can, however, be used to produce immune serum (NAGASHIMA 1958, KATZ et al. 1958, ARAKAWA et al. 1959, WARREN et al. 1961 , RILLEMAN et al. 1961).

II. Tissue culture a) Isolation of the measles virus PLOTZ (1938) propagated the virus in minced embryo tissue cultures with which he was able to induce measles in monkeys. RAKE (1941) obtained similar findings. However, the modern tissue culture technique which makes the quantitative and qualitative study of the virus possible was first used by ENDERS and PEEBLES (1954) for the isolation of virus using heparinized blood and throat washings collected from measles patients within 24 hours after appearance of the rash. They used human and Rhesus monkey kidney cell cultures. The presence of the virus is betrayed by the formation, through union of monolayer cells, of focal areas resembling syncytia or multinuclear giant cells in which large and small vacuoles are distributed throughout the cytoplasm, as weil as by slow destruction of the whole cell sheet. In stained preparations eosinophilic inclusion bodies are seen in the majority of nuclei enmeshed within the syncytial masses and cytoplasm. They were able to conduct complement fixation and neutralization tests with this virussuspensionmaterial as an antigen. Thereafter, the viruswas isolatcd by many workers: CoHEN et al. (1957), RucKLE and RooERS (1957) in primary human amnion cultures and human and monkey kidney, WRIGHT (1957) in monkey kidney, human amnion and chorion cultures, FRANKEL (1957) in monkey kidney and human renal cells, BECH and VON MAGNUS (1958) in monkey kidney and human amnion cultures, SMORODINTSEV et al. (1958) in human kidney, human cell cultures, etc., FRANKEL and WEST (1958), in FL cells, and MuTAI (1959) in monkey kidney cell cultures. ToYOSHIMA et al. (1959), YASUI (1960) and ARAKAWA (1962) in FL cells, SHINGU and NAKAGAWA (1960) in HeLa cells. Also ZHDANOV (1962) isolated USSR 58 from a patient's blood in the human amnion, monkey kidney and fibroblasts of chicken embryo in 1958. RucKLE (1957) states that the isolation of measles agents from throat secretion appears to be less successful in human amnion cell cultures than in human kidney cell cultures. On the other hand, when blood specimens are used, cytopathic changes appear after 3 days in human amnion cell cultures, and after 8 days in human kidney cultures, respectively. According to RucKLE and RoGERS (1957), the virus can be isolated, from blood and throat secretions, between 48 hours before and 32 hours after onset of the rash. They claim that there was a correlation between the presence of the virus in throat washings and blood

Recent advances in measles virology

9

specimens and the absence of neutralizing antihoclies in the serum. Their attempts, however, to isolate the measles virus from the blood and spinal fluids of patients with central nervaus symptoms were unsuccessful. FRANKEL (1957), on the other hand, obtained 4 positive spinal fluid specimens from patients with measles encephalitis, the specimens having been collected at a time when no virus was detectable in the blood or throat. FRANKEL (1957) reported positive fecal samples from several patients as late as 4-5 days after appearance of the rash, although Ruckle and Rogers obtained no virus from feces collected from 4 patients. GRESSER and KATZ (1960) isolated the virus from the urine of uncomplicated cases of measles. The majority of the specimens were collected within 48 hours after onset of rash, but occasionally the virus was detected 2 days after failure to demonstrate its presence in throat washings or blood. In smallpox, the virus may persist in the urine for a fairly long period (NAKAMURA and ÜFUJI 1925; NAKAMURA 1935). According to KIHOIN (1941), the variola virus is excreted in the urine of vaccinated subjects for 30 days after onset of illness, and for 40 days after onset of illness in the urine of non-vaccinated subjects.

b) Passage of the measles virus in tissue culture systems Epithelium like cells resembling human cancer cells are used in tissue cultures as the cellular lining vulnerable to the cytopathic effects of the isolated measles virus, notably giant cell formation. DEKKING and McCARTHY (1956) and BLACK et al. (1956) have observed cytopathic effects in KB cancer cell lining, and in HeLa and Hep-2 cells, respectively. Cytopathic effects have also been described by Jordan in human nasal mucosa cells (1956), by WRIGHT (1957) in the renal cells of the guinea pig, hamster and mouse, by WESTWOOD et al. (1957) in rabbit embryo kidney, by GIRARDI (1958) in human heart cell strains, by FRANKEL and WEST (1958) in stable human amnion FL cells, by FRANKEL et al. (1958) in dog's kidney cells, by ToYOSHIMA (1959) in human liver connective tissue cells, and by ScHWARZ and ZIRBEL (1959) and MATUMOTO et al. (1961) in bovine kidney cells. BERG and RosENTHAL (1961) were able to propagate the measles virus in suspensions of human and monkey leukocytes, and suggested that the virus might proliferate in polymorphonuclear cells. In reports on the cytopathic effects of the measles virus, not only multinuclear giant cell formation, but also fibroblast-like, spindie or stellate changes are described. RESSING et al. (1956), working with Hep-2 strain cells, have shown that giant cell formation is suppressed and spindie cell formation promoted by the addition of glutamine to the medium. MILOVANOVIC et al. (1957) described the formation of spindie cells in human amnion cell cultures, and FRANKELand WEST (1958) reported findings similar to those of RESSIG et al., namely that in FL cells giant cell formation was inhibited by the addition of glutamine. SELIGMAN and RAPP (1959) adduced an evidence to suggest that giant cell formation is peculiar to genetically distinct particles, and specific of t.he natural wild type of measles virus. The appearance of and transformation to spindie or stellate cells, however, is not absolute, and transmutationback to giant cells can occur even at limiting dilutions in 1 particular clone of Hep-2 cell Rtrain (RAPP 1960).

10

SEIJI

ARAKAwA :

Similar findings have been obtained with HeLa cells (Onno et al. 1961). MrTus (1962) isolated a strain of measles virus from the conjunctiva of a patient. Although this strain initially exhibited spindle-eeil formation, it is said that a stable variant producing only spindle-eeil transformation has not been isolated. These changes are likely to depend on virus strain, passage history, specific cell culture system, media temperature for different periods and different dilution etc., but their morphological significance is unknown (BLACK et al. 1959, McCARTHY 1962). In the Detroit-6 strain of human cells derived from human hone marrow, cytopathic changes in unstained cultures are apparently not readily recognized (ENDERS et al. 1957). KATZ et al. (1958) succeeded in propagating a meas1es virus strain adapted to chick amnion cells in chick embryo cells. In the first 4 serial passages in vitro cytopathic effects did not appear and in the 5th passage, for the first time there was rounding of some cells and formation of smaller multinucleated giant cells. Eosinophilic intranuclear and cytoplasmic inclusion bodies similar to those of the infected primate cells were also observed.

c) Attenuated virus and virulent virus ENDERS et al. (1959a) have utilized the egg adapted strain for the production of vaccine because of its lowered pathogenicity to monkeys and antigenicity comparable tothat of non-egg-adapted virus. BuYNAK et al. (1962) did a marker test on the non-egg-adapted and egg adapted strains, with the following results: In tissue culture, for instance, the egg adapted strain does not cause cytopathic changes in the WISH strain of human amnion cells, while non-egg-adapted strain fails to cause such changes in chick embryo cells. Egg adapted strain forms plaques in Cereopithecus aethiops monkey kidney and chick embryo cells, changes which are not produced by the non-egg-adapted strain. It is reported that in Cereopithecus aethiops monkeys no strain gives rise to any detectable illness (enanthema, significant pyrexia or leukopenia). The nonadapted-strain injected intrathalamically and intracisternally causes a moderate and predominantly mononuclear cell infiltration, cuffed vessels and demyelinisation, while the egg adapted strain failed to develop lesions beyond those attributable to the inoculation. MASON and TrTus (1962) reported as follows: In primary monolayer cultures of whole chick embryo, egg adapted virus induced formation of characteristic spindie cells. Infection of primary human amnion cells with attenuated virus yielded only islands of degeneration surrounded by cytoplasmic inclusions in intact cells, while virulent meas1es produced typical cytoplasmic and intranuclear inclusions along with multinucleated giant cells followed by destruction of these cells. In a stable line of human amnion, on the other hand, differences in cytopathology were less pronounced. Thus, differentiation between strains was most marked when chick embryo and primary human amnion cells stained with an uniline dye and indirect fluorescent antibody were used. Also the bovine adapted strain of MuTAI (1962) fails to induce cytopathic effects in the FL stable human amnion strain, but does so in monkey renal cells. Smorodintsev's strain grown on fibroblasts of the developing chick embryo does not proliferate in the respiratory tract on inhalation, but 1eads to the appear-

Recent advances in measles virology

11

ance of neutralizing and complement fixing antiboilies in the blood when injected by the intracutaneous or subcutaneous route, leaving a high degree of resistance to re-vaccination with the more active amniotic virus. Zhdanov's strain (1961) is stated to be less pathogenic compared with Enders' or Smorodintsev's strains although there had been only 4 passages in human amnion cells. The Toyoshima strain of ÜKUNO et al. although egg-adapted gives rise to multinuclear giant cells in the FL stable human amnion strain. H. TANIGUCHI (1959) was able to cultivate the mouse adapted strain in chick embryo, human embryo and pig embryo cells and saw multinuclear giant cells and cytoplasmic inclusion bodies in monkey renal cell culture. Enders' strain made by ARAKAWA (1962) 8 passages in eggs and 2 passages in mice was found to give rise to the characteristic cell degeneration in both primary human amnion cells and FL cells. According to ScHWARZ (1962), Enders' Edmonston strain after 77 passages in chick embryo cells exhibits little or no pathogenicity in man. This corresponds to the loss of pathogenicity to cows of the rinderpest virus following its repeated passagein developing chick eggs, as reported by NAKAMURA et al. (1953, 1954, 1957, 1958).

d) Spontaneous agents similar to the measles virus in uninoculated cultures The appearance in non-inoculated tissue cultures of monkey cells, of agents hardly distinguishable from the measles virus because of the similarity in their cytopathic effects and latent period, was described by RusTIGAN et al. (1955), RucKLE (1957) and BROWN (1957). Some of these agents can be identified serologically and by other properties, as measles virus (RucKLE 1956, BROWN 1957, RucKLE 1958, 1962). Besides these, however, an agent called the "foamy agent" occasionally appears which falls to give rise to intranuclear inclusions and also differs serologically.

e) Appearance of multinuclear giant cells ALAGNA (1911) found giant cells in the lymph nodes of tonsils of patients, during the 1908 epidemic in Palermo. LATER (1931), WARTHIN, and FINKELDEY, at the same time but independently, observed multinuclear giant cells in the tonsils and pharyngeal mucosa of patients in the prodromal stage (from 24 to 96 hours before appearance of the exanthema) and at the end of incubation and beginning of the prodromal stage, respectively. Next, the giant cells were seen by HERZBERG (1932) in lymphoid follicles of the appendix, and by GRÄFF (1937) in Rosenmüller's recess on the right side of the epipharynx, andin the pharynx, tonsils, the retropharyngeal and cervical lymph nodes, but not in the follicles of the appendix and spieen. He consequently considered the changes in the epipharynx and regional lymph nodes to be the primary complex of measles. HATHWAY (1935) found multinuclear giant cells in the spieen and scattered lymph nodes in the prodromal stage and MASUGI and MINAMI (1938) at autopsy of a child which had died on the 4th day of the exanthema, found giant cells of epithelial origin in the mucosa of bronchus, mouth, pharynx and epipharynx, as weil as eosinophilic inclusion bodies in the vicinity of nuclei. ToMPKINS and

12

SEIJI ARAKAWA:

MACAULEY (1955) observed multinuclear giant cells in the nasal secretions during the prodromal stage. Thus, in measles, the formation of reticuloendothelial and epithelial giant cells is the object of keen interest, representing specific cellular change. Since the work of ENDERS and PEEBLES (1954), the formation of giant cells in monolayer cultures has also been known to occur, and has become an accepted criterion for the presence of virus. RucKLE (1958) has isolated the virus from spieen, lung, kidney and lymph nodes of a patient who had died in the prodromal stage of measles in the absence of secondary infection. He found epithelial and reticulo-endothelial giant cells in these organs, and noted the presence of syncytial giant cells in the mucosa of the urinary bladder in one of three patients who had died after appearance of the rash. ENDERS et al. (1959) have isolated the measles virus from the body of a child who had died of giant cell pneumonia, thus demonstrating the aetiological relationship between the measles virus and Hecht's giant cell pneumonia (1910). AoYAMA {1959) has traced the chronological order of events in the formation of giant cells by means of the phase contrast microscope in infected monolayers. He found that the appearance of cytoplasmic inclusion bodies preceded the formation of giant cells, which takes place by the fusion of not only infected cells, but also of normal with infected cells. He stressed the close resemblance of these giant cells to Warthin-Finkeldey's reticulo-endothelial giant cells. ToYOSHIMA et al. (1960) have described the formation of giant cells in response to virus inactivated with ultra-violet light, and ScHLUEDERBERG (1962) has found, by means of the density gradient technique, also the non-infectious fraction with haemolytic activity tobe capable of inducing giant cell formation. The appearance of giant cells in monkeys has already been described, and reference made to the relationship between giant cells and spindie or stellate cells in monolayer cultures.

f) Inclusion bodies Since ToRRES and TEIXEIRA {1932) found the inclusion body in the stratum granulosum and stratum Malpighii of exanthema on the 4-6th day of measles, it has been described by many workers. Its presence in the monolayer system, has also been frequently mentioned since the work of ENDERS and PEEBLES. However, as stated by BLACK et al. (1959), the inclusion body is not an essential feature of the process of measles virus multiplication. Thus, in some system, such as human heart cellline, inclusion bodies have not been seen and the cytopathic effect of the virus is limited to elongation of the cells into spindie forms prior to their ultimate destruction. ÜOHEN et al. {1955) found the inclusion bodies, by fluorescent antibody technique, first in the perinuclear zone and later in the nuclei themselves in the form of masses. AoYAMA (1959) obtained similar findings employing the phase contrast microscope. The intranuclear inclusion is Feulgen negative (BLACK et al. 1959), and the cytoplasmic inclusion is susceptible to ribonuclease (TOYOSIDMA 1959). KALLMAN et al. (1959), BAKER et al. (1960), TAWARA et al. (1961), by means of the electron microscope, found the inclusion composed of filaments of high electron density, arranged in random manner. Based on a moreminnte electron-microscopic examination, TAWARA (1962) has advanced the view that the filamentaus fibers, and rod-like strands, or the parti-

Recent advances in measles virology

13

culate appearance in an ordered array or crystallike group as preciously described may conform to the initial helical component of about 170- 180 A, reported by W ATERSON et al. (1961 ).

111. Physical properlies of the measles virus BAKER et al. (1960) have described a particle about 1200 A in diameter on

the surface of cells infected with the virus. WATERSON et al. (1961), by means of the phosphotungstic acid negative contrast medium technique, have shown the particulate structure of the virus to be related to that of the myxoviruses. Thus, the overall diameter of most particles ranges from 1200-2500 A (Fig. 2a),

Fig. 2a. Measles virus (Micrography kindly supplied by Dr. WATERSON)

their structure generally resembling that of the Newcastle disease virus (HoRNE et al. 1960). The particles have an internal helical component of 170-180 A diameter with a central hole of about 50 A diameter, enclosed by an outer membrane (Fig. 2b). The filterability of this virus has been studied by GoLDBERG ER and ANDERSON (1911), DEGKWITZ (1927, 1927/28) and BLAKE and TRASK (1921), who observed its passage through the Berkefeld filter , and by RAKE and SHAFFER (1939/40) and ARAKAWA (1948) working with Seitz EK. Finding that the virus will pass through a gradocol membrane with an a.p.d. of 210 mfJ, but not through one with an a .p.d. of 190 m{J or less, BENYESH et al. (1958) have estimated the diameter of the particles to be about 140 mfJ . The ultrafiltration through gradocol membrane of mouse brain suspension in distilled water with 1 % normal guinea pig serum has been attempted by NAGASHIMA (1959) . He found that the virus passed through a membrane of 130 m{J a.p.d. with difficulty, and that its passage was almostimpossible through one of 114 m{J a.p.d., and quite impossible through one of 98 m{J a.p.d. (Table 1). The end point, thus, lies somewhat below 130 mfJ,

14

SEIJI ARAKAWA:

although this reading has been rejected as an exceptional case from their experimental results by BENEYSH et al. Working on the problern of stability, GoLDBERGER and ANDERSON (1911}, experimenting with monkeys, found that the virus was inactivated on heating to 55° C for 15 minutes. RAKE and SHAFFER (1940) found the virus to survive for several days at 0° C, and for 4 weeks at -35° C. According to HURST and CooKE (1941) virulence of lyophilized material was preserved for several months. ENDERS and PEEBLES (1954) reported destruction of tissue culture virus on heating to 65° C for 30 minutes, while heparinised blood or throat washings in milk remained infective to tissue culture for 31( 2 hours on refrigeration at 1-5° C.

Fig. 2b. Inner component of measles virus released from disrupted pa rticle (Micrography kindly supplied by

Dr. WATERSON)

In tissue culture fluids, the virus remained infective for 38 days at -15° C. MEUTHING et al. (1957) found Iosses of 2.0 and 1.2 log units after 1 month at 37 and 35° C, respectively, while RucKLE and RoGERS (1957) report a loss of 1.2log units at 36.5° C in 1 day, with a loss of most activity after 4 days and complete inactivation after 7 days. BLACK (1958) has performed accurate measurements with the aid of plaque counts on Hep-2 cell material at 0° C, and found no change at pH rauging from 6.0-10.5, at 0°C for 3 hours, but complete inactivation at pH values below 4.5. NAGASHIMA (1959) , working with a 10% saline emulsion of mouse brain at 37° C, was able to demoostrate progressively more rapid inactivation at pH values below 5, the suspension being most stable in the neighbourhood of pH 7. MussER and UNDERWOOD found the fall in titre to be small even after one year at -72° C. It is, however, comparatively unstable at 25 and 37° C, activity being nearly lost after 17-30 days, with a tailing off to complete inactivation in the subsequent period. At 25° C, stability is maximal at a pH of 7-8. Lyophilization may be carried out without apparent loss of titre. On ultracentrifuging for 1 hour at 8000 g, the supernatant fluid remained slightly infective, but the titre of the sediment was the same as that obtained

15

Recent advances in measles virology

on centrifuging for 1 hour at 127000 g, when all of the virus appears in the sediment. From these findings, the size of the virus was estimated to be about 140 mf1. Complete inactivation occurred with ß-irradiation in liquid medium between 3 X 105 and 5 X 105 , and with y irradiation at 2 X 105 rep, when the initial virus titre was 10 4-105 TCID50 fml. According to MARKHAM (1961), the titre of Enders live vaccine changes from log 5.5-3.5 to 0-1.2 on keeping at 4° C for 13-16 months, and from 6.2 to 2.2 on keeping at 37° C for one week. GoLDNER (1962) found that there Table l. Ultrafiltration test on mause adapted measles virus (NAGASHIMA, 1959) Average pore diameter (ml')

154

ÜHKI

I i

I 1 ,_, 1.5

10

1--- l.5

7

127 114

I

98

154

ÜHKI

127

,--· --···-

SHIBATA

I

Inoculation result*

I

~Tl

6

1 ,_, l.5

8

4 5 5 6 6 6 7 10 11 s

8

6 7 7 7 8

1 ,_, l.5

114

1 ,_, l.5

207

1 ,_, l.5

I

8 s s s s s s s s s s s s s s s

---

;

I

8

811 s s s s s s s s s s s s s

-------·

154

1 ,_, l.5 .

123

1 ,_, l.5 1 ,_, l.5

7

II

6 i... I

'

I

6

lO - 6

5

I

·-

----

6

'

* Numerals:

I0-4

I

1 ,_, 1.5

----

· - - -

114

LD,.

I

5 6 7 7 8 8 lO 12 12 13 - - - f - - - - - -------------1 ,_, l.5 8 8 g 10 12 8 10 13 14 s s

----~----

--·-

Amount of filtrate (ml)

t" F"lt 1 ra 100 pressnre

3 3 4 4 4 5 5 5 6 7 4 5 5 7 7 8 8 s s s 7 8 10 15 s

I0-4

s s s s s s s s s s s s s s s -·

-----

Day of occurrence of illness and death.

S Normal (Survival).

was no decline in titre at the end of 15 months at - 70° C. On lyophilization, the titre fell by log 0.5-0.75. There was complete inactivation after one month at 37° C, almost complete inactivation after 6 months at 25° C, while activity remained fairly stable for 11 months at 4° C. GIRARDI et al. (1958) reported reduction in complement fixing titre of tissue culture fluid on heating and ultraviolet irradiation, but resistance to exposure to formaldehyde for 13 days at 37° C. At 56° C the virus is inactivated in 60 minutes, as it is also by exposure to 2 cycles of ultraviolet light. According to NAGASHIMA (1959), a 10% saline suspension of brain material is inactivated completely within 3 days by 0.01-0.1% sodium ethylmercurithiosalicylate, and within 2 days by 0.01% formalin at 37° C.

16

SEIJI ARAKAWA:

ScHLUEDERBERG and ROIZMAN (1962) and ScHLUEDERBERG (1962) have demonstrated by means of equilibrium sedimentation that infectious measles virus culture fluids contain a large heterogeneaus population of specific measles virus antigenic particles differing from one another in respect of infectivity, haemagglutinating activity, haemolytic activity and density. The apparent buoyant density of the infectious particles was estimated to be approximately 1.29 gfml, and that of the two non-infectious complement fixing antigens 1.24 gjml and 1.14 gfml. There was, in particular, close similarity between the haemolytic and the complement fixing activity curves. The haemagglutinin curve, by contrast, differs considerably. DE MEIO, in addition, reports that the haemolytic activity of the virus declines on exposure to 56° C for 30 minutes while haemagglutination remained unaffected. YAGI (1961) have pointed out that the virus inactivated by sodium ethylthiosalicylate inhibits the proliferation of the measles virus, suggesting that interferon is being produced. This inhibiting substance can be made by inoculating embryonated hens egg, either in vivo or in vitro, with the inactivated virus. The production of interferon has actually been demonstrated by MAYER and ENDERS (1961). In view of the resistance of this virus to 3-bis(2-chloroethyl)-DL alanin (No. 243 derivative of nitrogen mustard), TAGAMI (1961) considers that the organism should be regarded as an RNA type virus, being distinguishable, by virtue of its inactivation with cholic acid and precipitation with protamine sulphate, from the arbor viruses, such as murine encephalo-myelitis (GD VII strain), murine myocarditis, and the polio and Coxsackie viruses.

IV. Relationship to other viruses As stated above, the viruses of canine distemper, rinderpest, and measles resemble one another closely both in their physico-chemical properties and their appearances on electron microscopy. They are considered to belong to the group of myxoviruses. PINKERTON et al. (1945) considered the relationship between measles and canine distemper with reference to the problern of giant cell pneumonia with inclusion bodies. CARLSTRÖM (1957) was the first to draw attention to the immunological relationship between the two viruses, as revealed by neutralisation tests of the canine distemper virus against convalescent measles serum. This has since been confirmed by cross reactions with various strains and by means of a variety of immunological methods, as reported by ADAMS and lMAGAWA (1957), ADAMS et al. (1958), ÜARLSTRÖM (1958), WARREN et al. (1956), BECH (1960), GILLESPIE et al. (1960), and SuzuKI et al. (1961). Comparison of certain physical properties of the distemper and measles viruses carried out and reported by PALM and BLACK (1961) supports the hypothesis that the two organisms belong to the same group. PLOWRIGHT et al. (1957), PoLDING and SIMPSON (1957), GoRET et al. (1957) and PoLDINGet al. (1959) have discussed the relationship between distemper and rinderpest, while GoRET et al., PLOWRIGHT et al., lMAWAGA et al. (1960), and WARREN et al. (1962) have established the relationship

17

Recent advances in measles virology

between measles, canine distemper and rinderpest by immunological means. However, as already pointed out, a number of workers have been unsuccessful in conferring protection against measles with this virus (ScHWARZ et al. 1960), HoEKENGA et al. (1960), while ÜABASSO et al. (1959, 1960) have even been unable to establish the occurrence of cross reactions.

V. Vaccine a) Prevention HERRMANN (1915) attempted active immunisation by the application to the nasopharyngeal mucosa of healthy infants within the first 5 months of life, of blood samples collected from measles patients during the 24 hours preceding the appearance of the rash. HIRAISHI and ÜKAMOTO (1921) also carried out prophylactic inoculation on 44 children, using diluted blood collected at the height of the rash, the diagnosis ha ving in most cases been confirmed by the presence of Koplik spots. The first vaccination against measles employing modern culture techniques was carried out by RAKE and coworkers, followed by STOKES et al. (1943), using chick embryo adapted virus. GELLIS et al. (1949) found that the majority of 479 subjects vaccinated developed mild symptoms. On subsequent exposure to infection, 330 (69%) contracted the disease, as compared with 44 (88%) of 50 children serving as controls. RITOSSA and MuL:E (1941) are said to have successfully induced a modified form of measles with chick embryo culture virus. TANIGUCHI et al. (1956) claim to have obtained excellent results by the nasal inoculation of live chick embryo adapted virus, in spite of the negligible symptoms in man. In view of the common antigenic properlies shared by the viruses of measles and canine distemper, ADAMS and IMAGAWA (1959) have attempted to confer immunity against measles with the aid of distemper vaccine. The contraction rate obtained was 1.8% as compared with one of 4.7-6.2% in a control group, but the significance of these results was not established. According to SCHWARZ et al. (1960) application of the distemper vaccine gave rise to the formation of antihoclies against distemper, but neutralising antihoclies against measles failed to develop. HoEKENGA et al. (1960) have reported the incidence of measles in the vaccinated group to be 14/388 against 25/414 in controls. The ENDERS' school have conducted an extensive series of prophylactic inoculations with egg adapted virus under controlled experimental conditions. The manufacture of most vaccines presently in use is based on their tissue culture technique. Enders' live vaccine gives rise to no immediate or local reaction at the site of injection, but the majority of subjects vaccinated developed measles in a mild forni.. Pyrexia Iasting for about 3 days sets in after about a week, with temperatures averaging 39.2° C per rectum. Temperatures in excess of 39.4° C were observed in about 20% of the subjects. In 45% a modified rash appears, usually on the 10th or 11th day after the temperature has settled. The exanthema is discrete, pink, and macular and does not proceed to desquamation. Distribution is variable, spreading from the postauricular region to cheeks, neck, or trunk, and in exceptional cases to involve the whole body. Only a few of the subjects complained of transient irritability, anorexia, cough, Ergebnisse der Mikrobiologie, Bd. 38

2

18

SEIJI ARAKAWA:

injected conjunctivae, coryza or abdominal discomfort, while Koplik spots were noted sporadically. Children with immunity exhibited no response to injection of the attenuated virus. The virus cannot be recovered from the blood, throat secretions or urine, by means of the usual techniques. Repeated and prolonged exposure to vaccinated siblings and roommates has failed to lead to either manifest or subclinical infection. KATZ et al. (1962) have carried out a 3 year follow up study of 95 vaccinated subjects, of which not a single one developed the illness as against 50-100% in control groups. At the end of 3 years there were positive serological responses in 96.7% of 311 children who had been free from antihoclies prior to inoculation. Titres obtained are said to be quantitatively comparable with those found after natural measles, and quantitatively the antihoclies are indistinguishable in vitro (BECH 1959, BLACK 1959). HoRNICK et al. (1962) were able to recover the virus from the blood and pharynx after inoculation with a vaccine made from canine renal or chick cell culture of an avianised strain, and observed somewhat stronger reactions than ENDERS et al. There was, however, no spread in susceptible populations. HALONEN et al. report similar clinical symptoms with pyrexia in 100%, and rashes in about 85% of which 50% were of the generalised maculo-papular type hardly distinguishable frommoderate natural measles. Of further practical interest are the reports of SMORODINTSEV (1958) and ZHDANOV (1962) that the reactions obtained by them were relatively mild, and that they are currently attempting to perfect a technique of 2 and 3 dose inoculations. ÜKUNO (1962) working with chick adapted virus and WAKO et al., and MINAGAWA et al. (1961) working with MATUMOTo's bovine kidney adapted virus received reactions very similar to those reported by KATZ et al. Simultaneously immunisation with viral antigens and gamma globulins has been attempted by McCRUMB et al., RILLEMAN et al. (1961-1962), and KRuGMAN et al. (1962) with the object of conferring immunity without the side effects of fever and rash. McCRUMB has studied the effect of simultaneous and contralateral injection by the intra-muscular route of 0.5-1.0 ml (10-1000 TCID50 (ml) and gamma globulin. He has found that in order to obtain satisfactory results, the vaccine should be of 100 TCID 50 or more, and that 12 neutralising units per pound body weight represent the minimal effective dose of gamma globulin. He adds that further work needs to be done to explore the antigen antibody relationship. RILLEMANN et al. are largely in agreement, but point out that little or no effect is to be expected when the two substances are given as one injection from the same syringe. They find that the combined use of vaccine and gamma globulin produces a situation similar to that found in infants, when passively acquired maternal antihoclies may suppress infection with live vaccine at a level no Ionger dernonstrahle by theserum neutralisation test (SToKES et al. 1960). In other words, infection through family exposure is suppressed even though the neutralising antibody titre is less than 1 : 1, 1: 2, 1:4 or so in vaccinated subjects receiving prophylactic inoculation (REILLY et al. 1961, STOKES et al. 1961, RILLEMAN et al. 1961 and 1962). KRUGMAN et al. (1962) carried out serial observations on measles antibody titres in 1700 subjects for one year, and found similar patterns after both vacci-

Recent advances in measles virology

19

nation and natural infection. With gamma-globulin modified vaccine, however, the antibody titre was lower. They also report a small outbreak of measles in a children ward, then 13 out of 18 (72%) presumably susceptible unvaccinated children contracted measles. Those who developed antihoclies in response to inoculation with live vaccine and gamma globulin failed to develop the illness, while those who produced no detectable antibodies were infected. In addition, the following findings are reported: a) a complement fixing antibody response of 65% was obtained with 80 TCID 50 +gamma globulin 0.02 mlfpound body weight (titre of gamma globulin is 1:400/0.1 ml against 100 TCID 50 ), b) a response of 96% with a vaccine of 1800 TCID 50 , and c) one of 98.6% with the same dosein combination with gamma globulin, 0.01 mlfpound body weight. However, pyrexia exceeding 101° F (38.3° C) did occur in 46% of the subjects treated as under b) andin 65% as under c). The advantage of the combined method is the less severe reaction, while the disadvantages are higher cost, and weaker immunity than that conferred by live vaccine alone. RILLEMAN et al. (1961, 1962) have conducted experiments with a formalin inactivated vaccine. This vaccine has, in fact, been used as control against live vaccine, but it has induced hardly any antigenic response in the human organism. A good antigenic response has been obtained experimentally with concentrated viral antigen treated with alum. According to WARREN and CuTCHINS (1962) serological changes occurred in 90-100% of subjects receiving 3 doses of the concentrated, formalin inactivated vaccine. During a three months follow-up period, WINKELSTEIN et al. (1962) found a morbidity of 20/350 subjects receiving 3 inoculations, as compared with 32/319 in controls. Among the vaccinated, there was less absenteeism from school, and sero-negative children tended to develop antibodies. Ethylene oxide inactivated vaccine has been found of promising by FRANKEL (1962). ARAKAWA (1949) has inoculated non-infected children with mouse adapted virus (- log LD 50 : about 5). Although pyrexia (37-38° C in the axilla), and measles like rashes over face, hands, extremities, back, and retro-auricular region were occasionally seen 8-12 days after the inoculation, almost no systemic reactions were present. As an example of the results, 106/142 (73%) of inoculated children living in a dormitory type residence remained uninfected, while only 6/68 (8%) in the control group escaped infection. By way of side effects, slight conjunctivitis was seen in 5/142 inoculated children, and a temperature of 37° C in the axilla was noted in two of them, one week after the inoculation. Of 21 susceptible school children aged 6-7 years, 81% were spared, while only 16% of 43 control children failed to contract the illness. In 1950, however, several workers reported that none of those developing exanthema were infected with measles or that the infection rate was exceedingly low in those with conjunctivitis, or that infection on exposure was completely inhibited. Unfortunately, we have not been able to conclude the effectiveness of this vaccine, owing to the contradictory results presented by some other workers. It was, however, thought that these results had been influenced by the inclusion of cases who became infected before vaccination was completed, and 2*

Inactivated Inactivated Inactivated

Inactivated Inactivated Inactivated

1 2 3

1 2 3

Inactivated Inactivated Inactivated

Live Live

Inactivated

Live

1 2 3

1 2

1

2

2

I Inactivated

II~~ctivated-

Inactivated Live

2 3

1

Live

1

-~--

Vaccine

Number ofinocu· lation

I

1000-1200